A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for material transport and provide mechanical support. The lateral roots (LRs) absorb sufficient water and nutrients. The genetic basis of vascular tissues and LRs development in rapeseed remains unknown. This study characterized an EMS-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced LRs, and leaf wilting. Scanning electron microscopy observations showed that the internode-cell shortened. Observations of the tissue sections revealed defects in the development of vascular bundles in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity confirmed that BnaA03.IAA13 is the functional gene, a G-to-A mutation in second exon changed the glycine at the 79th position to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID was conserved during plant evolution. The heterozygote of T16 significantly reduced the plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissues and LRs development, and provide a new germplasm resource for rapeseed breeding.

Developmental pleiotropy of SDP1 from seedling to mature stages in B. napus.

Rapeseed, an important oil crop, relies on robust seedling emergence for optimal yields. Seedling emergence in the field is vulnerable to various factors, among which inadequate self-supply of energy is crucial to limiting seedling growth in early stage. SUGAR-DEPENDENT1 (SDP1) initiates triacylglycerol (TAG) degradation, yet its detailed function has not been determined in B. napus. Here, we focused on the effects of plant growth during whole growth stages and energy mobilization during seedling establishment by mutation in BnSDP1. Protein sequence alignment and haplotypic analysis revealed the conservation of SDP1 among species, with a favorable haplotype enhancing oil content. Investigation of agronomic traits indicated bnsdp1 had a minor impact on vegetative growth and no obvious developmental defects when compared with wild type (WT) across growth stages. The seed oil content was improved by 2.0-2.37% in bnsdp1 lines, with slight reductions in silique length and seed number per silique. Furthermore, bnsdp1 resulted in lower seedling emergence, characterized by a shrunken hypocotyl and poor photosynthetic capacity in the early stages. Additionally, impaired seedling growth, especially in yellow seedlings, was not fully rescued in medium supplemented with exogenous sucrose. The limited lipid turnover in bnsdp1 was accompanied by induced amino acid degradation and PPDK-dependent gluconeogenesis pathway. Analysis of the metabolites in cotyledons revealed active amino acid metabolism and suppressed lipid degradation, consistent with the RNA-seq results. Finally, we proposed strategies for applying BnSDP1 in molecular breeding. Our study provides theoretical guidance for understanding trade-off between oil accumulation and seedling energy mobilization in B. napus.

Functional genomics of Brassica napus: Progresses, challenges, and perspectives.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

Corrigendum: Genome-wide analysis of transcriptome and histone modifications in Brassica napus hybrid.

[This corrects the article DOI: 10.3389/fpls.2023.1123729.].

Fine mapping of BnDM1-the gene regulating indeterminate inflorescence in Brassica napus.

KEY MESSAGE: A candidate gene Bndm1 related to determinate inflorescence was mapped to a 128-kb interval on C02 in Brassica napus. Brassica napus plants with determinate inflorescence exhibit improved traits in field production, such as lower plant height, improved lodging resistance, and consistent maturity. Compared to plants with indeterminate inflorescence, such features are favorable for mechanized harvesting techniques. Here, using a natural mutant 6138 with determinate inflorescence, it is demonstrated that determinate inflorescence reduces plant height significantly without affecting thousand-grain weight and yield per plant. Determinacy was regulated by a single recessive gene, Bndm1. Using a combination of SNP arrays and map-based cloning, we mapped the locus of determinacy to a 128-kb region on C02. Based on sequence comparisons and the reported functions of candidate genes in this region, we predicted BnaC02.knu (a homolog of KNU in Arabidopsis) as a possible candidate gene of Bndm1 for controlling determinate inflorescence. We found a 623-bp deletion in a region upstream of the KNU promoter in the mutant. This deletion led to the significant overexpression of BnaC02.knu in the mutant compared to that in the ZS11 line. The correlation between this deletion and determinate inflorescence was examined in natural populations. The results indicated that the deletion affected the normal transcription of BnaC02.knu in the plants with determinate inflorescence and played an important role in maintaining flower development. This study presents as a new material for optimizing plant architecture and breeding novel canola varieties suitable for mechanized production. Moreover, our findings provide a theoretical basis for analyzing the molecular mechanisms underlying the formation of determinate inflorescence in B. napus.

Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea).

Biosynthesis, stabilization, and storage of carotenoids are vital processes in plants that collectively contribute to the vibrant colors observed in flowers and fruits. Despite its importance, the carotenoid storage pathway remains poorly understood and lacks thorough characterization. We identified two homologous genes, BjA02.PC1 and BjB04.PC2, belonging to the esterase/lipase/thioesterase (ELT) family of acyltransferases. We showed that BjPCs in association with fibrillin gene BjFBN1b control the stable storage of carotenoids in yellow flowers of Brassica juncea. Through genetic, high-resolution mass spectrometry and transmission electron microscopy analyses, we demonstrated that both BjA02.PC1 and BjB04.PC2 can promote the accumulation of esterified xanthophylls, facilitating the formation of carotenoid-enriched plastoglobules (PGs) and ultimately producing yellow pigments in flowers. The elimination of BjPCs led to the redirection of metabolic flux from xanthophyll ester biosynthesis to lipid biosynthesis, resulting in white flowers for B. juncea. Moreover, we genetically verified the function of two fibrillin genes, BjA01.FBN1b and BjB05.FBN1b, in mediating PG formation and demonstrated that xanthophyll esters must be deposited in PGs for stable storage. These findings identified a previously unknown carotenoid storage pathway that is regulated by BjPCs and BjFBN1b, while offering unique opportunities for improving the stability, deposition, and bioavailability of carotenoids.

A systematic dissection in oilseed rape provides insights into the genetic architecture and molecular mechanism of yield heterosis.

Heterosis refers to the better performance of cross progeny compared with inbred parents, and its utilization contributes greatly to agricultural production. Several hypotheses have been proposed to explain heterosis mainly including dominance, over-dominance (or pseudo-overdominance) and epistasis. However, systematic dissection and verification of these hypotheses are rarely documented. Here, comparison of heterosis level across different traits showed that the strong heterosis of composite traits (such as yield) could be attributed to the multiplicative effects of moderate heterosis of component traits, whether at the genome or locus level. Yield heterosis was regulated by a complex trait-QTL network that was characterized by obvious centre-periphery structure, hub QTL, complex up/downstream and positive/negative feedback relationships. More importantly, we showed that better-parent heterosis on yield could be produced in a cross of two near-isogenic lines by the pyramiding and complementation of two major heterotic QTL showing partial-dominance on yield components. The causal gene (BnaA9.CYP78A9) of QC14 was identified, and its heterotic effect results from the heterozygous status of a CACTA-like transposable element in its upstream regulatory region, which led to partial dominance at expression and auxin levels, thus resulting in non-additive expression of downstream responsive genes involved in cell cycle and proliferation, eventually leading to the heterosis of cell number. Taken together, the results at the phenotypic, genetic and molecular levels were highly consistent, which demonstrated that the pyramiding effect of heterotic QTL and the multiplicative effect of individual component traits could well explain substantial parts of yield heterosis in oilseed rape. These results provide in-depth insights into the genetic architecture and molecular mechanism of yield heterosis.

Integrating biochemical and anatomical characterizations with transcriptome analysis to dissect superior stem strength of ZS11 (Brassica napus).

Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

The transcription factor BnaWRKY10 regulates cytokinin dehydrogenase BnaCKX2 to control cytokinin distribution and seed size in Brassica napus.

Cytokinins (CKs) are phytohormones that promote cell division and differentiation. However, the regulation of CK distribution and homeostasis in Brassica napus is poorly understood. Here, the endogenous CKs were first quantified by LC-ESI-MS/MS in rapeseed tissues and visualized by TCSn::GUS reporter lines. Interestingly, the cytokinin oxidase/dehydrogenase BnaCKX2 homologs were mainly expressed in reproductive organs. Subsequently, the quadruple mutants of the four BnaCKX2 homologs were generated. Endogenous CKs were increased in the seeds of the BnaCKX2 quadruple mutants, resulting in a significantly reduced seed size. In contrast, overexpression of BnaA9.CKX2 resulted in larger seeds, probably by delaying endosperm cellularization. Furthermore, the transcription factor BnaC6.WRKY10b, but not BnaC6.WRKY10a, positively regulated BnaA9.CKX2 expression by binding directly to its promoter region. Overexpression of BnaC6.WRKY10b rather than BnaC6.WRKY10a resulted in lower concentration of CKs and larger seeds by activating BnaA9.CKX2 expression, indicating that the functional differentiation of BnaWRKY10 homologs might have occurred during B. napus evolution or domestication. Notably, the haploid types of BnaA9.CKX2 were associated with 1000-seed weight in the natural B. napus population. Overall, the study reveals the distribution of CKs in B. napus tissues, and shows that BnaWRKY10-mediated BnaCKX2 expression is essential for seed size regulation, providing promising targets for oil crop improvement.

Genome-wide analysis of transcriptome and histone modifications in Brassica napus hybrid.

Although utilization of heterosis has largely improved the yield of many crops worldwide, the underlying molecular mechanism of heterosis, particularly for allopolyploids, remains unclear. Here, we compared epigenome and transcriptome data of an elite hybrid and its parental lines in three assessed tissues (seedling, flower bud, and silique) to explore their contribution to heterosis in allopolyploid B. napus. Transcriptome analysis illustrated that a small proportion of non-additive genes in the hybrid compared with its parents, as well as parental expression level dominance, might have a significant effect on heterosis. We identified histone modification (H3K4me3 and H3K27me3) variation between the parents and hybrid, most of which resulted from the differences between parents. H3K4me3 variations were positively correlated with gene expression differences among the hybrid and its parents. Furthermore, H3K4me3 and H3K27me3 were rather stable in hybridization and were mainly inherited additively in the B. napus hybrid. Together, our data revealed that transcriptome reprogramming and histone modification remodeling in the hybrid could serve as valuable resources for better understanding heterosis in allopolyploid crops.

miR319 and its target TCP4 involved in plant architecture regulation in Brassica napus.

Plant architecture is a collection of genetically controlled crop productivity and adaptation. MicroRNAs (miRNAs) have been proved to function in various biological processes, but little is known about how miRNA regulates plant architecture in rapeseed (Brassica napus L.). In this study, four small RNA libraries and two degradome libraries from shoot apex of normal and rod-like plants were sequenced. A total of 639 miRNA precursors and 16 differentially expressed miRNAs were identified in this study. In addition, 322 targets were identified through degradome sequencing. Among them, 14 targets were further validated via RNA ligase-mediated 5' rapid amplification of cDNA ends. Transgenic approach showed that increased TCP4 activity in Arabidopsis resulted in premature onset of maturation and reduced plant size along with early flowering and shortened flowering time. miR319-OE lines in Brassica napus exhibited serrated leaves and abnormal development of shoot apical meristem (SAM), which led to the deformed growth of stem and reduced plant height. In conclusion, our study lays the foundation for elucidating miRNA regulate plant architecture and provides new insight into the miR319/TCP4 module regulates plant architecture in rapeseed.

Kinase CIPK9 integrates glucose and abscisic acid signaling to regulate seed oil metabolism in rapeseed.

Rapeseed (Brassica napus), an important oil crop worldwide, provides large amounts of lipids for human requirements. Calcineurin B-like (CBL)-interacting protein kinase 9 (CIPK9) was reported to regulate seed oil content in the plant. Here, we generated gene-silenced lines through RNA interference biotechnology and loss-of-function mutant bnacipk9 using CRISPR/Cas9 to further study BnaCIPK9 functions in the seed oil metabolism of rapeseeds. We discovered that compared with wild-type (WT) lines, gene-silenced and bnacipk9 lines had substantially different oil contents and fatty acid compositions: seed oil content was improved by 3%-5% and 1%-6% in bnacipk9 lines and gene-silenced lines, respectively; both lines were with increased levels of monounsaturated fatty acids and decreased levels of polyunsaturated fatty acids. Additionally, hormone and glucose content analyses revealed that compared with WT lines the bnacipk9 lines showed significant differences: in bnacipk9 seeds, indoleacetic acid and abscisic acid (ABA) levels were higher; glucose and sucrose contents were higher with a higher hexose-to-sucrose ratio in bnacipk9 mid-to-late maturation development seeds. Furthermore, the bnacipk9 was less sensitive to glucose and ABA than the WT according to stomatal aperture regulation assays and the expression levels of genes involved in glucose and ABA regulating pathways in rapeseeds. Notably, in Arabidopsis (Arabidopsis thaliana), exogenous ABA and glucose imposed on developing seeds revealed the effects of ABA and glucose signaling on seed oil accumulation. Altogether, our results strongly suggest a role of CIPK9 in mediating the interaction between glucose flux and ABA hormone signaling to regulate seed oil metabolism in rapeseed.

Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L.

Brassica napus L. (canola, oil seed rape) is one of the world's most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants' vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study's findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

Comparison of dynamic 3D chromatin architecture uncovers heterosis for leaf size in Brassica napus.

INTRODUCTION: Heterosis is the major event driving plant development and promoting crop breeding, but the molecular bases for this phenomenon remain elusive. OBJECTIVES: We aim to explore the effect of three-dimensional (3D) chromatin architecture on the underlying mechanism of heterosis. METHODS: Here, we constructed the North Carolina II (NC-II) population to select superior and inferior heterosis sets by comparing mid-parent heterosis (MPH) in Brassica napus. To decipher the impact of 3D chromatin architecture on the underlying mechanism of heterosis, we combined genetics, transcriptomics and 3D genomics approaches. RESULTS: We suggest that F1 hybrids with superior heterosis tend to contain more transcriptionally active A compartments compared with F1 hybrids with inferior heterosis, and approximately 19-21% compartment significantly altered in the F1 hybrids relative to the parental lines. Further analyses show that chromatin compartments correlate with genetic variance among parents, which may form the basis for differentially active chromatin compartments. Having more A compartments in F1 hybrids confers a more accessible chromatin circumstance, which promotes a higher proportion of highly expressed ELD (expression level dominance) genes in superior heterosis F1 hybrids (46-64%) compared with inferior heterosis F1 hybrids (22-31%). Moreover, genes related to hormones which affect plant growth, are more up-regulated with changes of 3D genome architecture, and we validate that increased hormone content contributes to cell proliferation and expansion by influencing the key genes of cell cycle thereby promoting leaf size. CONCLUSION: Dynamic 3D chromatin architecture correlates with genetic variance among parents and contributes to heterosis in Brassica napus.

CRISPR/Cas9-Mediated Targeted Mutagenesis of BnaCOL9 Advances the Flowering Time of Brassica napus L.

Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. The planting area and output of rapeseed are affected by the flowering time, which is a critical agronomic feature. COL9 controls growth and development in many different plant species as a member of the zinc finger transcription factor family. However, BnaCOL9 in rapeseed has not been documented. The aim of this study was to apply CRISPR/Cas9 technology to create an early-flowering germplasm resource to provide useful material for improving the early-maturing breeding of rapeseed. We identified four COL9 homologs in rapeseed that were distributed on chromosomes A05, C05, A03, and C03. We successfully created quadruple BnaCOL9 mutations in rapeseed using the CRISPR/Cas9 platform. The quadruple mutants of BnaCOL9 flowered earlier than the wild-type. On the other hand, the flowering time of the BnaCOL9 overexpression lines was delayed. An analysis of the expression patterns revealed that these genes were substantially expressed in the leaves and flowers. A subcellular localization experiment demonstrated that BnaCOL9 was in the nucleus. Furthermore, we discovered that two key flowering-related genes, BnaCO and BnaFT, were highly elevated in the BnaCOL9 mutants, but dramatically downregulated in the BnaCOL9 overexpression lines. Our findings demonstrate that BnaCOL9 is a significant flowering inhibitor in rapeseed and may be employed as a crucial gene for early-maturing breeding.

Genome-wide detection of genotype environment interactions for flowering time in Brassica napus.

Flowering time is strongly related to the environment, while the genotype-by-environment interaction study for flowering time is lacking in Brassica napus. Here, a total of 11,700,689 single nucleotide polymorphisms in 490 B. napus accessions were used to associate with the flowering time and related climatic index in eight environments using a compressed variance-component mixed model, 3VmrMLM. As a result, 19 stable main-effect quantitative trait nucleotides (QTNs) and 32 QTN-by-environment interactions (QEIs) for flowering time were detected. Four windows of daily average temperature and precipitation were found to be climatic factors highly correlated with flowering time. Ten main-effect QTNs were found to be associated with these flowering-time-related climatic indexes. Using differentially expressed gene (DEG) analysis in semi-winter and spring oilseed rapes, 5,850 and 5,511 DEGs were found to be significantly expressed before and after vernalization. Twelve and 14 DEGs, including 7 and 9 known homologs in Arabidopsis, were found to be candidate genes for stable QTNs and QEIs for flowering time, respectively. Five DEGs were found to be candidate genes for main-effect QTNs for flowering-time-related climatic index. These candidate genes, such as BnaFLCs, BnaFTs, BnaA02.VIN3, and BnaC09.PRR7, were further validated by the haplotype, selective sweep, and co-expression networks analysis. The candidate genes identified in this study will be helpful to breed B. napus varieties adapted to particular environments with optimized flowering time.

The dynamics of lncRNAs transcription in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization is the intrinsic forces behind genome evolution. Long non-coding RNAs (lncRNAs) are important for plant biological processes regulation. However, it is unclear that these non-coding fractions are impacted by interspecific hybridization. Here we examined the profiles of lncRNAs by comparing them with coding genes in Brassica napus, three accessions of Brassica rapa, and their F1 hybrids. 6206 high-confidential lncRNAs were identified in F 1 hybrids and their parentals, and the lncRNAs transcriptome in the F1 hybrids was reprogrammed by the genome shock. Notably, genome-wide unbalanced of lncRNAs were observed between An and Ar subgenomes, ELD (Expression Level Dominance) was biased toward the An -genome in F1 hybrids, and ELD of non-conserved lncRNAs was more than conserved lncRNAs. Our findings demonstrate that the reprogramed lncRNAs acts as important role in enhancing plant plasticity, leading to the acquisition of desirable traits in polyploid Brassica species.

Transcription factor WRKY28 curbs WRKY33-mediated resistance to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia sclerotiorum causes substantial damage and loss of yield in oilseed rape (Brassica napus). The molecular mechanisms of oilseed rape defense against Sclerotinia remain elusive. In this study, we found that in the early stages of B. napus infection a conserved mitogen-activated protein kinase (MAPK) cascade mediated by BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module phosphorylates the substrate BnWRKY33, enhancing its transcriptional activity. The activated BnWRKY33 binds to its own promoter and triggers a transcriptional burst of BnWRKY33, thus helping plants effectively resist the pathogenic fungi by enhancing the expression of phytoalexin synthesis-related genes. The expression of BnWRKY33 is fine-tuned during defense. Ongoing Sclerotinia infection induces BnaA03.WRKY28 and BnaA09.VQ12 expression. BnaA09.VQ12 interacts physically with BnaA03.WRKY28 to form a protein complex, causing BnaA03.WRKY28 to outcompete BnWRKY33 and bind to the BnWRKY33 promoter. BnaA03.WRKY28 induction suppresses BnWRKY33 expression in the later stages of infection but promotes branch formation in the leaf axils by regulating the expression of branching-related genes such as BnBRC1. BnaA03.WRKY28 participates in the trade-off between defense and growth. These findings suggest that oilseed rape plants may modulate defense-response strength and develop alternative reproduction and survival strategies in the face of lethal pathogens.

Bulk segregant analysis-sequencing and RNA-Seq analyses reveal candidate genes associated with albino phenotype in Brassica napus.

Inheritable albino mutants are excellent models for exploring the mechanism of chloroplast biogenesis and development. However, only a few non-lethal albino mutations have been reported to date in Brassica species. Here, we describe a resynthesized Brassica napus mutant, whose leaf, stem, and silique tissues showed an inheritable albino phenotype under field conditions after the bud stage but green phenotype in the greenhouse during the whole growing season, indicating that the albino phenotype depends on environmental conditions. Compared with the green leaves of the field-grown wild-type (GL) and greenhouse-grown mutant (WGL) plants, white leaves of the field-grown mutant (WL) showed significantly lower chlorophyll contents and structural defects in chloroplasts. Genetic analysis revealed that the albino phenotype of WL is recessive and is controlled by multiple genes. Bulk segregant analysis-sequencing (BSA-Seq) indicated that the candidate regions responsible for the albino phenotype spanned a total physical distance of approximately 49.68 Mb on chromosomes A03, A07, A08, C03, C04, C06, and C07. To gain insights into the molecular mechanisms that control chloroplast development in B. napus, we performed transcriptome (RNA-Seq) analysis of GL, WGL, and WL samples. GO and KEGG enrichment analyses suggested that differentially expressed genes (DEGs) associated with leaf color were significantly enriched in photosynthesis, ribosome biogenesis and chlorophyll metabolism. Further analysis indicated that DEGs involved in chloroplast development and chlorophyll metabolism were likely the main factors responsible for the albino phenotype in B. napus. A total of 59 DEGs were screened in the candidate regions, and four DEGs (BnaC03G0522600NO, BnaC07G0481600NO, BnaC07G0497800NO, and BnaA08G0016300NO) were identified as the most likely candidates responsible for the albino phenotype. Altogether, this study provides clues for elucidating the molecular mechanisms underlying chloroplast development in B. napus.